Solid Phase Extraction (SPE) is a very simple technique to use, employing disposable extraction columns or microplates (see FIG. 1) which are available in a wide range of reservoir volumes, formats and sorbents. In principle, SPE is analogous to liquid-liquid extraction (LLE). As a liquid sample passes through the SPE column, compounds are ‘extracted’ from the sample and adsorbed onto the support or sorbent material in the column. Interferences can then be selectively removed from the column using the correct choice of wash or interference elution solvents. Finally, the desired analytes may be selectively recovered from the column by an elution solvent, resulting in a highly purified extract. The analyte concentration in this extract is often higher than in the original sample.
Alternatively, an extraction column may be selected that retains the interferences present in the sample, but allows the analytes to pass through un-retained, providing clean-up but not analyte trace enrichment. SPE sorbents have a typical mean particle size of 30-50 μm. Many organic solvents can flow through SPE columns or plates under gravity, but for aqueous samples and more viscous solvents, liquids must be passed through the sorbent bed using vacuum applied to the column outlet, positive pressure applied to the column inlet, or centrifugation (see FIG. 2).
The Supported Liquid Extraction (SLE) process is analogous to traditional liquid-liquid extraction (LLE) and utilizes the same water immiscible solvent systems for analyte extraction. However, instead of shaking the two immiscible phases together, the aqueous phase is immobilized on an inert diatomaceous earth based support material and the water immiscible organic phase flows through the support, alleviating many of the liquid handling issues associated with traditional LLE such as emulsion formation. As a result recoveries are often higher and demonstrate better reproducibility from sample to sample.
In sample preparation, the principles of traditional LLE (partitioning of analytes between aqueous and water immiscible organic solvents) are well known and understood. Traditionally, analytes are extracted from aqueous samples through the addition of an appropriate water immiscible organic solvent. The two immiscible phases are shaken or mixed thoroughly in a separating funnel, and based on relative solubility of the analytes in the two phases, analytes will partition into the organic solvent. The efficiency of the extraction is enhanced by the shaking, which creates a high surface area for the extraction interface allowing partitioning to occur.
Liquid-liquid extraction can give particularly clean extracts of biological fluids, since matrix components such as proteins and phospholipids are not soluble in typical LLE solvents, and are therefore excluded from the final extract. The same benefits are true for supported liquid extraction (SLE) procedures.
Because the same water immiscible solvents are used in SLE, proteins and phospholipids are efficiently removed from the final extract, and no additional steps such as protein crash (precipitation) are required. Using a fast, simple load-wait-elute procedure, SLE provides inherently cleaner extracts than other simple sample preparation techniques such as ‘dilute and shoot’ or protein precipitation. The efficient extraction process combining high analyte recoveries, elimination of emulsion formation, and complete removal of matrix interferences such as proteins, phospholipids, and salts results in lower limits of quantitation compared to traditional LLE.
ISOLUTE® SLE+ products from Biotage (Uppsala, Sweden) contain a modified form of diatomaceous earth, which provides a support for the liquid-liquid extraction process to occur, but does not interact chemically with the aqueous sample. Application of the sample to the column results in the aqueous sample spreading over the surface of the material, forming an immobilized layer of small droplets held in place by a network of pores (FIG. 3). When the water immiscible extraction solvent is applied for the elution step, it flows over the aqueous droplets allowing efficient analyte partitioning. The large surface area of the extraction interface and flow through nature of the technique leads to a very efficient extraction procedure, because analytes come into contact with fresh solvent as the organic phase travels through the bed, mimicking a repeat LLE mechanism.
Processing SLE columns and 96-well plates is largely performed under gravity, with a pulse of vacuum or positive pressure used to initiate loading of the sample, and to maximize solvent recovery (leading to more reproducible analyte recovery) after elution. Both manual and automated, vacuum or positive pressure systems can be used.
A recommended workflow for processing Biotage's ISOLUTE SLE+ columns and plates is:
1. Pre-treat sample as required (including internal standard addition)
2. Ensure appropriate collection vessel is in place
3. Load sample onto ISOLUTE SLE+ column or plate
4. Apply vacuum (−0.2 bar) or pressure (3 psi) for 2-5 seconds to initiate loading
5. Wait 5 minutes for sample to completely absorb and form extraction layer
6. Apply water immiscible extraction solvent and allow to flow for 5 minutes under gravity
7. Apply vacuum (−0.2 bar) or pressure (10 psi) for 10-30 seconds to complete elution
8. Evaporate eluate to dryness and reconstitute as required.
Automated systems for transferring liquid samples between sample containers and sample processing containers are commercially available. Such systems are available e.g. from Perkin Elmer under the tradename “Janus”, from Tecan Trading under the tradename “Freedom EVO”, and from Tomtec under the tradename “Quadra”.
Such systems generally comprise (i) a sample container for holding a sample, (ii) a solvent container for holding a solvent, (iii) a sample processing container in the form of a column or a 96-well plate comprising a sample processing material, and (iv) a liquid handling robot arranged to move an aliquot of the liquid sample from the sample container to the sample processing container, and also to move an aliquot of the solvent from the solvent container to the sample processing container.